Human cytomegalovirus (HCMV) is a common pathogen which generally causes subclinical infection but can cause serious disease in immunocompromised individuals - e.g., cancer, AIDS, and bone marrow transplant patients. The long term goal of this project is to develop an understanding of the molecular mechanisms involved in HCMV latency and reactivation. In an effort to identify cellular reservoirs of HCMV in the peripheral blood, we have found that HCMV seropositive individuals harbor virus in circulating peripheral blood mononuclear (PBMN) cells. He will extend these studies to determine in vitro the ability of HCMV immediate-early (IE). early (E), and late (L) genes to express in lymphocytes in culture, PBMN cells, and PBMN subset populations. We will also examine the ability of HCMV to replicate in endothelial cells, another potential latent site of the virus. Representative HCMV genes of the three kinetic classes will be assayed for production of steady- state RNA by in vitro nuclear runoff transcription of nuclei from infected cells. In addition, IE, E. and L promoters will be examined for their ability to function in transiently transfected cells. To determine the extent of HCMV replication in endothelial cells in vivo, arterial specimens will be examined by in situ hybridization and immunocytochemistry with nucleic acid probes and antibodies representing IE, E, and L genes. The final part of the proposal will identify essential cis elements in the regulatory region of the major IE gene and regulatory proteins which interact with this sequence in nonpermissive teratocarcinoma cells and permissive cells. Using teratocarcinoma cells, we have identified a potentially important cis regulatory element in the 5' flanking sequence of the IE gene. We will examine this element and other sequences in the regulatory region by gel retardation assays and DNaseI footprint analysis. We will use an in vitro transcription assay system to analyze the importance of these bound sequences. We will also construct point mutations in the IE regulatory region and analyze by transient transfection. Finally we will insert mutated sequences back into the virus by homologous recombination to test the importance of these cis sequences in vivo. Results from the proposed experiments will advance our knowledge of mechanisms involved in HCMV latency and reactivation as well as transcription in general.